44] Radioimmunoassay of the major plasma metabolite of PGF2α, 15-keto-13,14-dihydro-PGF2α

Abstract

Publisher Summary This chapter discusses radioimmunoassay of the major plasma metabolite of PGF2α, 15-Keto-13,14-dihydro-PGF2α. In this particular assay, plasma volumes of as much as 0.5 ml may be assayed without disturbing interferences from other plasma constituents. After the addition of the labeled ligand, the tubes are left for incubation. The next step is the separation of the free and the antibody-bound fractions. Of all methods tried, 3 the polyethylene glycol method gave the most reliable results. The tubes are vigorously vortexed, the formed grayish-white pellet contains the precipitated γ-globulins including the antibody-bound fraction of the prostaglandin metabolite, whereas the free fraction is found in the supernatant. The yellow color of unextracted plasma gives about 10% reduction in counting efficiency in the scintillation counter as compared to the standard vials without plasma. It is important that the counted values for counts per minute are corrected for quenching. This can easily be done if the scintillation counter is equipped with automatic quench correction facilities, such as automatic external standard channels ratio. Before setting up routine radioimmunoassay runs, the optimal titer of the antibody must be established. This should always be done also with the commercial preparations, as experience shows that the recommended procedure from the manufacturer is often far from optimal.